The cartilage proteoglycan aggregate is largely composed of complexes of hyaluronic acid and proteoglycans which are maintained and stabilized by the so called Link protein (LP). LP contains several distinct functional domains that specifically bind protein and hyaluronic acid. There are two adjacent tandemly repeated hyaluronic acid binding domains (commonly called the proteoglycan tandem repeats, or PTR, domains) in LP. Although the solution structure of an ancestral PTR domain (TSG-6) has been reported, the details of its interaction with HA and metal ions are largely uncharacterized. No structure of the LP PTR domains have been reported and their interactions with HA and metal ions are not fully characterized. The long range goal of this program is, therefore, to determine the structures of both PTR domains from bovine (and human) link protein. It is anticipated that in addition to a three dimensional solution structure, that information on the conformation effects of metal ion and hyaluronic acid binding will be obtained and that the protein residues involved will be identified. The goals of this feasibility project are to express, as recombinant proteins in E. coli and insect cell expression system, the approximately 100 residue PTR domains for ligand binding and NMR structural analysis. Specifically we will: 1) express recombinant bovine PTR-1, PTR-2 and the joined PTR1-PTR2 domains and optimize their (re)folding; 2) characterize and quantitate their metal and HA binding properties; and 3) obtain optimal NMR solution conditions and begin the detailed NMR structural analysis by high field multidimensional proton NMR spectroscopy. Initial attempts to locate the HA and metal ion binding sites of these proteins will also be made. These studies will eventually lead to the understanding of the molecular/ atomic details of the hyaluronic acid-PTR domain interactions which may be useful for the rational design of therapeutics that could ultimately enhance cartilage homeostasis. The results of this work will also be directly applicable to the study of similar PTR domains in the aggrecan and versican molecules which form a ternary complex with the link protein and HA.